Project summary One of the most important goals of dentistry and the NIDCR/NIH is to generate a whole ?Bio- Tooth?. Dentin is a critical structural component of tooth. Although it has made great progress of dentin development benefit for numerous hereditary syndromes and chromosomal anomalies, many gaps have been remained. Understanding signaling pathways of dental mesenchymal lineages and dentin formation would provide an avenue for repair and regeneration of dentin. Dentin sialophosphoprotein (Dspp) is highly expressed in odontoblasts and dentin and processed in to dentin sialoprotein (Dsp) and dentin phosphoprotein (Dpp). Dsp and Dpp mutations are associated with dentinogenesis imperfecta (DGI), which is the most common dentin genetic disorder. Our long-ranged goal is to elucidate the biological mechanisms controlling dentin formation, which will fill a key gap of knowledge leading to dentin regenerating. The objective here is to define the signaling pathways essential for dentinogenesis. Our central hypothesis is that the novel control mechanisms, in which the several signals among Bmp2-pAkt-pErk-Dlx3-Sp7-Gcn5-Dspp as well as Dsp-integrin ?6 and Dsp-occludin play synergic roles in controlling dentin formation. The hypothesis is based on our strong preliminary data produced using both global knockout (KO) and conditional KO (cKO) mouse models as well as in vitro cellular and molecule approaches. Three Specific Aims are proposed to test this hypothesis: 1). to determine Bmp2 signaling in Dspp expression and dentin formation. Bmp2 signal plays functional roles: Dspp expression via up- regulation of Bmp2-pAkt-Erk-Dlx3-Sp7-Gcn5G signaling pathways. 2). to rescue dentin formation in Bmp2 KO mice by overexpression of Dspp gene. Due to dramatically decrease of Dspp expression and dentin defect in Bmp2 KO mice, the hypothesis is that Dspp overexpression in Bmp2 KO mice is able to rescue dentin formation. 3). to determine Dsp signaling in dental mesenchymal cell differentiation and dentin formation. Dsp is processed by MMP9 into active fragments, which as ligands bind to cell membrane receptors, integrin ?6 and occludin. The Dsp-?6 complex positively stimulates Dspp expression and odontoblast homeostasis through up-regulation of Smad1/5/8 signaling. Dsp-occludin signal enhances phosphorylation of focal adhesion kinase (FAK), promoting dental mesenchymal cell differentiation. The proposed research is innovative because each step of transcription, posttranslational processing and signaling transduction of Dsp/Dspp is necessary for the formation of healthy dentin. Such knowledge will advance our understanding of the pathogenesis of inherited disorders that threaten the structural integrity of dentin and provide a potential clue for treating dental diseases and dentin regeneration.